Chenodeoxycholic Acid-Amikacin Combination Enhances Eradication of Staphylococcus aureus

ABSTRACT The rise of antibiotic resistance and dearth of novel antibiotics have posed a serious health crisis worldwide. In this study, we screened a combination of antibiotics and nonantibiotics providing a viable strategy to solve this issue by broadening the antimicrobial spectrum. We found that chenodeoxycholic acid (CDCA), a cholic acid derivative of the traditional Chinese medicine (TCM) Tanreqing (TRQ), synergizes with amikacin against Staphylococcus aureus in vitro, and this synergistic killing was effective against diverse methicillin-resistant S. aureus (MRSA) variants, including small-colony variants (SCVs), biofilm strains, and persisters. The CDCA-amikacin combination protects a mouse model from S. aureus infections. Mechanistically, CDCA increases the uptake of aminoglycosides in a proton motive force-dependent manner by dissipating the chemical potential and potentiates reactive oxygen species (ROS) generation by inhibiting superoxide dismutase activity. This work highlights the potential use of TCM components in treating S. aureus-associated infections and extend the use of aminoglycosides in eradicating Gram-positive pathogens. IMPORTANCE Multidrug resistance (MDR) is spreading globally with increasing speed. The search for new antibiotics is one of the key strategies in the fight against MDR. Antibiotic resistance breakers that may or may not have direct antibacterial action and can either be coadministered or conjugated with other antibiotics are being studied. To better expand the antibacterial spectrum of certain antibiotics, we identified one component from a traditional Chinese medicine, Tanreqing (TRQ), that increased the activity of aminoglycosides. We found that this so-called agent, chenodeoxycholic acid (CDCA), sensitizes Staphylococcus aureus to aminoglycoside killing and protects a mouse model from S. aureus infections. CDCA increases the uptake of aminoglycosides in a proton motive force-dependent manner by dissipating the chemical potential and potentiates ROS generation by inhibiting superoxide dismutase activity in S. aureus. Our work highlights the potential use of TCM or its effective components, such as CDCA, in treating antibiotic resistance-associated infections.

concentration required for inhibiting growth of S. aureus it results rather toxic for mammalian cells (<20% survival rate; Figure  S4). The authors, however, have identified an interesting synergy between CDCA and the aminoglycoside amikacin ( Figure 2), with many positive features: the combination of CDCA and amikacin prevents selection of mutants resistant to amikacin ( Figure  3B), it is active against variants of S. aureus (Figure 4), and it is active in an in vivo model of skin infection ( Figure 5), among other characteristics. Unfortunately, amikacin is not among the first line drugs for the treatment of staphylococcal infections, so the clinical relevance of this work is rather low.
The rationale for selecting amikacin as a companion drug to CDCA is not shown (line 133: We focus on amikacin...), so I would suggest to screen whether CDCA also presents synergy with other, more relevant drugs from the clinical point of view. If so, this would increase notably the clinical interest of this work.
In addition, some parts of the manuscript are unclear. For example, the use of certain control drugs or experimental conditions in various experiments should be better explained in order to have a clear idea about the meaning of the outcome of the experiment (for example, in figure 5, what is the difference between model and control?). Also, some sentences are vague, for example, line 252, ...restoring H concentration, where?, in line 175, ...repress the development of detectable mutations, TO AMIKACIN should be added for clarity. It would be good to review all the manuscript for providing enough details for a better comprehension. In lines 229-230, when it says that the increase of membrane permeability caused by CDCA IS NOT COMPARABLE to that of the increase of aminoglycoside accumulation, the rationale behind this assessment should be detailed.
Finally, the size of some figures is too small, thus preventing from proper analysis of data presented.
Reviewer #2 (Comments for the Author): The authors describe the in-depth characterization of a component of from the traditional Chinese medicine Tanreqing. They have identified this agent synergizes with aminoglycosides to kill Staphylococcus aureus. The agent, chenodeoxycholic acid dissipates the proton motor force without disruption of the membrane. The activity against persister development initiated by aminoglycosides is compelling research for future therapeutics. The detailed work describing the physiological effects of chenodeoxycholic acid revealed the mechanism used to synergize with aminoglycosides. With the importance of the findings of this work, minor modifications to the manuscript would greatly benefit readability and the understanding of this important synergistic abilities of chenodeoxycholic acid (Please, see additional comments in the attached file).

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The authors describe the in-depth characterization of a component of from the traditional Chinese medicine Tanreqing. They have identified this agent synergizes with aminoglycosides to kill Staphylococcus aureus. The agent, chenodeoxycholic acid dissipates the proton motor force without disruption of the membrane. The activity against persister development initiated by aminoglycosides is compelling research for future therapeutics. The detailed work describing the physiological effects of chenodeoxycholic acid revealed the mechanism used to synergize with aminoglycosides. With the importance of the findings of this work, minor modifications to the manuscript would greatly benefit readability and the understanding of this important synergistic abilities of chenodeoxycholic acid.
Major comments: 1. FICI values are commonly reported by adding the FICs of both drugs (∑FIC= FICantibiotic + FICcdca) and a value of < 0.5 is synergistic. Although the fact that these agents have synergistic activities is clear, only the FIC of the antibiotics are reported. It would be easier if the reviewer did not have to figure out the FIC of CDCA when tested with the antibiotics. 2. The biofilm work in figure 4 shows great promise for CDCAA. However, the effect of amikacin alone or combined with CDCA is not discussed in the text for its ability to disrupt a biofilm or any combinatorial effect seen or not seen with these experiments.

Reviewer#1:
The manuscript by Cui and co-workers have extensively characterized the antimicrobial activity of chenodeoxycholic acid (CDCA, a component of traditional Chinese medicines) against Staphylococcus aureus. This compound does not select for resistant mutants in vitro ( Figure 3A), but shows a rather low antimicrobial activity (MIC 320 µg/ml; Figure 1A) and at the concentration required for inhibiting growth of S. aureus it results rather toxic for mammalian cells (<20% survival rate; Figure S4). The authors, however, have identified an interesting synergy between CDCA and the aminoglycoside amikacin ( Figure 2), with many positive features: the combination of CDCA and amikacin prevents selection of mutants resistant to amikacin ( Figure 3B), it is active against variants of S. aureus (Figure 4), and it is active in an in vivo model of skin infection ( Figure 5), among other characteristics. Unfortunately, amikacin is not among the first line drugs for the treatment of staphylococcal infections, so the clinical relevance of this work is rather low. In addition, we found that CDCA as an active component of the TRQ had synergistic effects with various clinical antibiotics, including aminoglycosides and β-lactams. The synergistic mechanism of CDCA and aminoglycosides is distinct from other antibiotics, and the combination mechanism of other kinds of antibiotics is being studied successively. By using amikacin as a representative type of aminoglycosides, we aim to highlight the potential of CDCA as an adjuvant in expanding the antibacterial spectrum of AMK and other aminoglycosides.
In addition, we have changed the wording in the text and highlighted them as follows: "Previously, we have shown that TRQ combined with vancomycin or linezolid exhibited synergy against MRSA (30). In this study, we selected aminoglycoside class antibiotics to investigate their activity against Gram-positive pathogens. As shown in Fig. 2A

Response:
We thank the reviewer for pointing out this issue. We have changed the words of "augmenting ∆ψ" instead of "restoring H+ concentration". In Figure 6F, we used CCCP as a control to collapse PMF and more precisely, ∆ψ, and to prevent aminoglycoside uptake. However, CDCA could antagonized this activity by augment ∆ψ ( Figure 6E) and CDCAA finally sensitizes PMF-depleted bacterial populations to the killing of aminoglycosides.
4. in line 175, ...repress the development of detectable mutations, TO AMIKACIN should be added for clarity.
Response: Thanks for the comments. We have changed the words " repress the development of detectable mutations" to "repress the development of aminoglycoside resistance". Since we have noticed that we did not examine the mutations elicited by serial passages of bacteria. However, we indeed measured the MIC of these mutants and thus we used "resistance" instead of "mutations" in the revised text. However, it would be interesting to investigate the mutations underlying these mutants.
5. It would be good to review all the manuscript for providing enough details for a better comprehension. In lines 229-230, when it says that the increase of membrane permeability caused by CDCA IS NOT COMPARABLE to that of the increase of aminoglycoside accumulation, the rationale behind this assessment should be detailed.

Revised Fig. 6B Response:
Thanks for the critical review and constructive advice. We have updated the methods and results of Figure 6B to be consistent with the conditions in Figure 6A. We found that CDCA increased the cell membrane permeability by about 1.16-fold at the concentration of CDCA (40 μg/ml) as compared to the control group. However, the uptake of aminoglycosides increased 6.68-fold compared with control.
In addition, we have modified the results part as follows: "Interestingly, we noticed the increase of membrane permeability caused by CDCA is not comparable to that of the increase of aminoglycoside accumulation. We found that CDCA increased the cell membrane permeability by about 1.16-fold at the concentration of CDCA (40 μg/ml) as compared to the control group. However, the uptake of aminoglycosides increased about 6.68-fold compared with control. We therefore reasoned that other mechanisms might exist.". 6. Finally, the size of some figures is too small, thus preventing from proper analysis of data presented.
Response: Thanks for the critical review and constructive advice. We have redrawn the figures to make it clear and updated the legend accordingly in the revised manuscript.

Reviewer #2 (Comments for the Author):
The authors describe the in-depth characterization of a component of from the traditional Chinese medicine Tanreqing. They have identified this agent synergizes with aminoglycosides to kill Staphylococcus aureus. The agent, chenodeoxycholic acid dissipates the proton motor force without disruption of the membrane. The activity against persister development initiated by aminoglycosides is compelling research for future therapeutics. The detailed work describing the physiological effects of chenodeoxycholic acid revealed the mechanism used to synergize with aminoglycosides. With the importance of the findings of this work, minor modifications to the manuscript would greatly benefit readability and the understanding of this important synergistic abilities of chenodeoxycholic acid.
1. FICI values are commonly reported by adding the FICs of both drugs (∑FIC= FICantibiotic + FICcdca) and a value of < 0.5 is synergistic. Although the fact that these agents have synergistic activities is clear, only the FIC of the antibiotics are reported. It would be easier if the reviewer did not have to figure out the FIC of CDCA when tested with the antibiotics.
Response: Thank you for your kind suggestion. The FIC values we described in the article are all ∑FIC, which represents various antibiotics in combination with CDCA, but not FIC antibiotic . Therefore we draw a schematic to better demonstrate the content in the FIC heat map. And all heat maps showed the FIC antibiotic and the FIC CDCA , respectively and FICI could be calculated with the function of FICI = ∑FIC = FIC antibiotic + FIC CDCA = (MIC of antibiotic in combination/MIC antibiotic alone) + (MIC CDCA in combination/MIC CDCA alone). figure 4 shows great promise for CDCAA. However, the effect of amikacin alone or combined with CDCA is not discussed in the text for its ability to disrupt a biofilm or any combinatorial effect seen or not seen with these experiments.

Response:
We thank the reviewer for pointing out this issue. We found that bacteria could survive in the biofilm even at high concentrations in AMK (green fluorescence). And we suggest that while inhibiting the effect of the bacterial biofilm was attributed by CDCA, the red fluorescence with deepening concentration represents the effect of the drug combination. In addition, we added Figure S2 of the separate green fluorescence (live bacteria) channels to the supplementary data to support this conclusion.  Figure 2D is clear: the main text says that this corresponds to MSSA, the figure legend says MSSA ATCC 25923, and experiments have been done with 0.5 µg/ml amikacin, which is 0.5 x MIC of the MSSA strain. However, Figure 2E is really unclear, because the main text says that this corresponds to MRSA, the figure legend says MRSA ATCC 25923 (please note that this is the ATCC number for the MSSA strain; that for the MRSA strain is 43300), and experiments have been done with 10 µg/ml amikacin, which is 10 x MIC of the MSSA strain (why? 10 µg/ml would correspond to 0.15 x MIC of the MRSA strain). So, it looks as if both figures have done with the SAME strain (the MSSA strain only) but using two different concentrations of amikacin (0.5 and 10 µg/ml, being respectively 0.5 and 10 times the MIC of the MSSA strain). If so, then the text should be revised because references to antimicrobial activity of amikacin+CDCA against the MRSA strain should be changed to MSSA strain (for example, in lines 33, 152, 155, 156, 161, 163, 187, and probably others along the text). 2) Figure 4B, the scale bar is not shown 3) Figure 4D, what is the meaning of 'normal cells'? does it mean normal size? How has been determined? How many cells were inspected? 4) Legend to figure 5A, the sentence 'Model group means' is uncomplete 5) Figure 5, it is difficult to observe the black triangle. Please, try to increase contrast by adding a white line, using another color, or any other way. 6) Figure 6G, legend and figure do not correspond, legend only explains partially the content of the figure. 7) Figure 7A, change 'promoted' to increased or up-regulated, and 'repressed' to decreased or down-regulated Staff Comments:

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Reviewer comments:
Reviewer #1 (Comments for the Author): The authors have improved their manuscript in comparison with the original submission, although there are still a few points that need further work, mainly related with results and figures: 1) Figure 2D is clear: the main text says that this corresponds to MSSA, the figure legend says MSSA ATCC 25923, and experiments have been done with 0.5 µg/ml amikacin, which is 0.5 x MIC of the MSSA strain. However, Figure 2E is really unclear, because the main text says that this corresponds to MRSA, the figure legend says MRSA ATCC 25923 (please note that this is the ATCC number for the MSSA strain; that for the MRSA strain is 43300), and experiments have been done with 10 µg/ml amikacin, which is 10 x MIC of the MSSA strain (why? 10 µg/ml would correspond to 0.15 x MIC of the MRSA strain). So, it looks as if both figures have done with the SAME strain (the MSSA strain only) but using two different concentrations of amikacin (0.5 and 10 µg/ml, being respectively 0.5 and 10 times the MIC of the MSSA strain). If so, then the text should be revised because references to antimicrobial activity of amikacin+CDCA against the MRSA strain should be changed to MSSA strain (for example, in lines 33, 152, 155, 156, 161, 163, 187, and probably others along the text).

Response:
We thank the reviewer for pointing out this issue. We have changed the strain name in Figure 2E from "MSSA ATCC 25923" to "MRSA ATCC 43300" and corrected the MIC. To be sure of this, we thoroughly examined the "MSSA" and "MRSA" throughout the paper and are sure that the descriptions of strains in the main text are correct.